Fractionation of mixtures of proteinaceous substances using polyethylene glycol



United States Patent 3,415,804 FRACTIONATION OF MIXTURES 0F PRO- TEINACEOUS SUBSTANCES USING POLY- ETHYLENE GLYCOL Alfred Polson, Milnerton, Republic of South Africa, assignor to South African Inventions Development Corporation, Pretoria, Transvaal, Republic of South Africa, a corporation of the Republic of South Africa No Drawing. Continuation-impart of application Ser. No. 246,000, Dec. 20, 1962. This application Mar. 31, 1967, Ser. No. 627,308 Claims priority, application Republic of South Africa, Jan. 1, 1962, 34/62 13 Claims. (Cl. 260-112) ABSTRACT OF THE DISCLOSURE Mixtures of proteinaceous substances such as plasma proteins, toxins, antitoxins, enzymes, desoxyribonuclease and viruses are fractionated in aqueous medium with polyethylene glycol used as a dispersibility depressant resulting in a single liquid phase containing one fraction in dispersion and a solid phase fraction of a composition different from that of the original mixture.

CROSS-REFERENCES TO RELATED APPLICATIONS This is a continuation-in-part of patent application Ser. No. 246,000, filed Dec. 20, 1962, now abandoned.

BACKGROUND OF THE INVENTION The present invention relates to the fractionation of mixtures of proteinaceous substances such as plasma proteins, toxins, antitoxins, enzymes, desoxyribonuclease and viruses.

For separating the components of protein and related mixtures the alcohol fractionation method has hitherto been the most practical commercial method for achieving a comparatively large number of separations with reasonable sharpness. Salting out is also employed very commonly although the sharpness of the attainable separations is usually very poor. The use of ion exchangers is only practical on a laboratory scale. Ether fractionation is very inconvenient because of the fire hazard. Electrodecantation is inconvenient for fractionating complex mixtures on a large scale.

In the alcohol fractionation method various low molecular weight alcohols are employed. The most commonly used alcohol is ethanol although the use of low molecular weight monomeric glycols has also been reported, though Without any details and it appears that ethanol .has hitherto been considered the most convenient substance to use. In the alcohol fractionation method it is necessary to rely on large concentration increments to produce "fractionated precipitation. It must be carried out at very low temperatures, usually at least at 0 C., more often at even lower temperature, say down to C., to avoid denaturing the protein. This requires refrigeration and inconvenient operating conditions. Furthermore it severely limits the temperature range in which the process can be carried out. This limitation is serious because temperature variation is one of the factors suitable for achieving fractionation in the mixture. The disadvantage of a limited temperature range can sometimes be partly overcome by varying the pH of the solution being treated. On the other hand it is often necessary to operate in a limited pH range, e.g., in acid media (provided the proteins involved are stable at low pHs) where it is necessary to inhibit the digestion of the proteins by enzymes which may be present and which are active in alkaline media.

In the context of the above prior art it is one of the objects of the invention to provide a process suitable as an alternative to the alcohol fractionation method, which in many instances offers considerable advantages over the alcohol fractionation method and which can sometimes be applied when the alcohol fractionation method is unsuit- .able.

It is a further object to provide a process which in its preferred forms may be carried out at least for a major part thereof at room or incubator temperature and which is suitable to be carried out in an exceptionally large temperature range, say from approximately 40 C. (in some cases higher) down to the freezing point of the mixture to be treated.

It is another object of the invention to provide an industrial process, which has a greatly reduced tendency to denature, some proteins being treated under conditions which are known to favour denaturing when using the alcohol fractionating process, as will be explained more fully below.

Albertsson in Partition of Cell Particles and Macromolecules, 1960, Stockholm and New York (John Wiley), describes and discusses various liquid polymer phase systems wherein two liquid phases are formed by water and two polymers, one of which may be polyethylene glycol. These liquid two phase systems. are suggested and investigated for the separation of proteins by liquid-liquid partition, in particular using the countercurrent distribution techniques of Craig and coworkers. The work aims specifically at getting away from precipitation methods (page 10, first paragraph) because there is a greater chance of obtaining a state of equilibrium between two liquid phases than between a solid phase and a liquid phase, for example. This is particularly so when macromolecules are involved.

Albertsson suggests that polyethylene glycol has a stabilizing effect on proteins, inhibiting denaturation. He also demonstrates that in certain of his systems a precipitate forms at the interface between the two liquid phases, ascribed in part to surface tension elfects and in some instances at least in part to a reduced dispersibility. Since in each case two polymers are present in both liquid phases, it is not possible to ascribe the precipitation to any single one of the polymers. Nor is it, for example, ap parent from that literature that such polymers, for example polyethylene glycol alone, have any pronounced selectivity 'When precipitating proteins from a mixture dispersed in a single liquid phase.

In the context of Albertssons book, the occurrence of a precipitate is a disadvantageous limiting factor since it renders unfeasible any normal countercurrent distribution method. The object of the book is to describe the distribution of the largest number of macromolecules without precipitation (page 147, lines 15-16).

The liquid-liquid distributions described are of considerable scientific interest but have serious limitations and disadvantages when applied commercially. The interface is difiicult to detect and the phase separation is exceedingly slow (Va-1 hr. under favourable circumstances). Commercially interesting separations may require hundreds of transfers, equivalent to hundreds of hours (cf. Biochimica et Biophysica Acta, 69 (1963), 263-270).

Liquid-liquid partitions require large volumes of phases to ensure suflicient dilution for complete solubility. The separation must then be followed by the additional step of recovering the fractions from the liquids.

The present invention is based on the discovery of outstanding advantages attainable when polyethylene glycol is used to depress the dispersibility of proteinaceous substances of a mixture in the presence of a single liquid, more particularly aqueous, phase. Some of the most important advantages are the following (in addition to the advantages discussed further above in the context of the alcohol fractionation method) (a) Polyethylene glycol has an extreme selectivity as a dispersibility depressant when employed as will be described further below.

(b) It allows separations in between about one and three simple precipitation and/or selective redispersion steps which would require hundreds of transfers by the liquidliquid partition method.

(c) The disadvantages of precipitation methods suggested by Albertsson are not observed.

(d) The precipitation (or its reversal) is substantially independent of the concentration of the particular protein and is substantially only dependent on the concentration of the polyethylene glycol.

(e) The amount of substrate precipitated is not stoichiometrically dependent on the amount of precipitant. (f) The process allows the easy isolation of such substances as fibrinogen, which cannot be isolated satisfactorily by any liquid-liquid partition method.

(g) The process is highly adaptable.

SUMMARY OF THE INVENTION The process in accordance with the invention for fractionating a mixture of proteinaceous substances comprises:

(a) Bringing said mixture in finely divided form into admixture with water dispersible polyethylene glycol having a molecular weight substantially not less than 300 and substantially not more than 100,000, there being present also water in which said polyethylene glycol after said admixture is in a condition of dispersion ranging from true solution to colloidal dispersion;

(b) Adjusting the relative concentration of the proteinaceous substances on the one hand and the remaining components in the product resulting from said admixture on the other hand to a predetermined value in dependancy of pH and temperature at which value a part of said mixture is rendered indispersible in the water by the glycol, resulting in a fiocculated solid phase containing part of said mixture and a single liquid phase containing water and dispersed therein the remainder of said mixture with a composition different from the initial composition of said mixture;

(c) Separating said flocculated solid phase as one fraction from said single liquid phase as a second fraction; and

(d) Recovering a proteinaceous fractionation product of said mixture from at least one of said fractions. Polyethylene glycol (PEG) having a molecular weight in the range of 300 to 100,000 was found suitable. A

pronounced improvement is obtainable if the PEG has a molecular weight of at least 600 with an upper limit of 20,000. The best results were attainable with PEG having a molecular weight in the range 1,500 to 20,000, say

As a rule it is preferred to carry out the process with PEG having a limited molecular weight range, more particularly composed of molecules, the molecular weights of which are essentially within a narrow range by comparison with the total range as specified further above. However, it is sometimes possible to operate with mixtures of quite ditferent molecular weights.

It has been confirmed for the present process as well that polyethylene glycol acts as a stabiliser of protein solutions, a factor which is of particular importance in dealing with dilute solutions of protein, e.g., purified bacterial toxins which tend to denature on standing for prolonged periods.

The process may be carried out by fractional precipitation, for example at least two fractions are precipitated successively and selectively by increasing the concentration of the precipitant in the solution.

However, the reverse procedure is also possible, which then comprises the steps of fractionally extracting a solid mixture of the two or more proteinaceous compounds with an aqueous dispersion of PEG capable of selectively dispersing one of the said compounds while leaving the other compound at least partly undispersed. This may also be done by precipitating a mixture of at least two substances to be separated from one another and selectively eluting precipitated substance from the precipitate with at least one dispersion of PEG having a lower PEG concentration than the PEG concentration necessary to achieve complete precipitation.

It is also envisaged to carry out complex fractionations by repeated alternate precipitation and dissolution. Consecutive precipitations may be carried out under different conditions, e.g., with regard to the molecular weight of the PEG or by varying other factors as will be explained further below. It is also possible to combine the process with other fractionating processes, e.g., electrodecantation and alcohol precipitation, particularly in cases where any one method alone produces inadequate separations.

One feature observed in many applications of the process is the comparatively large change in precipitating power produced by comparatively small increments in the concentration of the PEG under otherwise constant conditions. As a result, it is often possible to separate a comparatively large number of different fractions merely by varying the PEG concentration. In addition other factors such as temperature, pH and ionic concentrations may be varied to further improve the fractionation.

For example, it is well known in other processes that a protein fraction is more readily precipitated from solution when the pH of the solution is close to the isoelectric point of the protein components to be removed. Also, it is known in other processes that protein fractions are more easily precipitated when the ionic concentration of the medium in which they are dispersed is lowered. Likewise the temperature may be varied to increase the separation between two protein fractions. The above-mentioned variations of pH, ionic strength and temperature have to some extent been found to have the same effect when applied to the present process.

In the case of the present process it is found, however, that of all three variables the effect of pH is by far the most noticeable. It is therefore particularly advantageous if the selectivity of the precipitation or redispersion is at least partly controlled by the control of the pH during precipitation.

The wide temperature range over which the process can be carried out represents a great advantage. Thus, it is possible to an extraordinary degree to adapt the process by a suitable choice of molecular weight of the PEG and if necessary by slightly modifying the concentration range of the PEG to a specific set of conditions with regard to pH, ionic strength and temperature desirable for any reasons whatsoever. Where conditions require a protein solution to have a low ionic concentration, this can be achieved by first precipitating all the protein and then redissolving the protein in a solvent having the required ionic concentration, thus avoiding the need to work at high dilutions.

Polyethylene glycol used in the process can be removed by extraction with a solvent, e.g., chloroform, provided such solvent does not denature the particular precipitate.

Eleetrodecantation may also be employed to remove small amounts of PEG from protein fractions. In other cases the removal of the PEG is unnecessary, in some cases the presence of the precipitant may even be desirable because of its stabilizing effect. Other possible methods of removing PEG from contaminated fractions involve the use of anion and cation exchangers, acetone or sometimes ammonium sulphate, alternatively, the protein or like fraction precipitated can be redispersed and then reprecipitated by salting-out procedures and with ethanol. However, it must be stressed once again for some applications the retention of minor amounts of PEG in the final product is not objectionable.

In general the interaction between the precipitant and the solution is brought about at a temperature essentially above 0 C., advantageously at a temperature at least as high as ordinary room temperature. In this respect the process differs in an important and very advantageous manner from the cold alcohol fractionation process. Another important difference is to be found in the feature that the best precipitates are obtained if the predetermined ration of PEG and solution of substance to be precipitated is brought into admixture within a period of minutes. For example, to obtain the best results the precipitant is admixed at a rate of at least 4 kg. per minute per 50 litres of solution. The precipitant may also be added to the solution cocurrently. By way of contrast the cold alcohol precipitation method requires the alcohol concentration to be raised to the required level over a much longer period. This difference is due to the fact that in the present process the interaction between PEG and substance being precipitated is usually completely reversible.

The precipitates formed by the process are on the whole particularly easy to handle, particularly if the solution after the admixture of the precipitating agent is maintained under conditions of gentle agitation before separating the precipitate from the solutions, say for a period of half an hour.

Some embodiments of the process applied to the fractionation of a mixture of compounds comprising the said protein type structure comprise carrying out the fractionation with at least two different precipitating agents in combination, of which at least one is an inorganic precipitant, capable as such of precipitating protein type substances from aqueous solution. Preferably, said inorganic precipitant is ammonium sulphate.

A particular application of the process to the separation of gamma-globulin from blood plasma comprises precipitating an impure gamma-globulin from plasma by the addition thereto of ammonium sulphate, dissolving the precipitate in an aqueous medium and mixing the solution with polyethylene glycol to recover a more purified precipitate of gamma-globulin. The last-mentioned precipitate is preferably redissolved in an aqueous medium, contaminants of the gamma-globulin are precipitated by mixing the solution with a predetermined amount of polyethylene glycol, the precipitate is separated from the remaining solution and the concentration of polyethylene glycol in the solution is incretased to precipitate gammaglobulin. Preferably the removal of impurities at the lower concentration of polyethylene glycol takes place essentially at a pH of 4.4-4.8 whereas the last-mentioned precipitation of the gamma-globulin takes place at a pH between 6 and neutral. The final precipitate of gamma-globulin resulting from precipitation with polyethylene glycol may be freed of polyethylene glycol contamination by redissolving the precipitate and precipitating the gammaglobulin from the solution with a different precipitant, e.g. cold ethanol in a manner known per se.

Another embodiment is applied to the separation of both fibrinogen and gamma-globulin and comprises mixing the plasma with ammonium sulphate up to a concentration at which most of the fibrinogen present precipitates, leaving the bulk of the gamma-globulin and some fibrinogen in solution, separating the precipitate from the solution, increasing the concentration of ammonium sulphate to precipitate at least the bulk of the gamma globulin and redissolving both precipitates separately, mixing the fibrinogemenriched solution with polyethylene glycol to precipitate an essentially pure fibrinogen fraction, while maintaining essentially neutral conditions and mix ingthe gamma-globulin-enriched solution with polyethylene glycol to precipitate therefrom gamma-globulin in a more highly purified form than before. The lastmentioned precipitate of gamma-globulin is preferably redissolved in an aqueous medium and further processed which was suitable for the in the manner described in the previous embodiment for the purification of gamma-globulin.

Experiments have indicated that the process can be used for the fractionation or purification of nucleic acids and live virus.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The following examples of specific and preferred embodiments can be modified and adapted to suit different problems by following the more general teachings under the heading Summary of the invention.

Example 1.Fractionation of human blood plasma Human plasma was diluted with water in a ratio of 1:1 to result in a solution having a protein concentration of 3.8 gms. per ml. water. 5 ml. portions of this solution were added to 5 ml. portions of PEG (molecular weight 6,000) of increasing concentration dissolved in 4 M phosphate buffer, pH 7.0. The mixtures were left in a water bath at 21 C. for 30 minutes and then spun down at this temperature at 14,000 rpm. for 15 minutes. Protein determinations were made of the supernatant fluids and these were plotted against the PEG concentrations.

The protein fraction which was precipitated at 3% was shown to be a fibrinogen concentrate as it clotted on addition of thrombin. At 8% PEG concentration the gamma-globulin fraction was recovered and between 21% and 26% PEG concentration the albumin fraction, contaminated with a trace of beta-globulin, was separated.

It was also shown that at lower temperatures, fractionation occurred at lower concentrations of PEG and that,

therefore, it was possible to fractionate with PEG by merely lowering the temperature.

In addition, it was shown that fractionation is more readily achieved at low protein concentrations and that precipitation proceeds more readily at low salt concentrations.

Thus it was shown that perfect separation in a single separation stage could only be achieved at total protein concentrations of less than 0.4 gm. per 100 ml. Under those conditions fibrinogen precipitated at between 0 and 4% PEG, gamma-globulin between 4 and 8%, betaglobulin between 8 and 12% and alpha-1 and alpha-2- globulins and albumins at appreciably higher than 12% PEG. Using higher protein concentrations it was found that there would be a certain overlap of the fractions.

It was also found that the optimum temperature for achieving selective precipitation was room temperature, i.e., about 20 C. It was furthermore found that of the different possible variables pH variations had a particularly striking influence on the selectivity of the precipitation. Thus it was found that complete separation of gamma-globulins from fibrinogen could only be effected at a pH between 4.4 and 4.8, preferably 4.6 (a pH value production of gamma-globulins of high quality, but which had an adverse effect on the fibrinogen). At pH 5.8 gamma-globulin and beta-globulin could be separated completely.

Example 2.-Purification of Clostridium novyi toxin 0.925 gm. of dried toxin was dissolved in 100 ml. of M phosphate buffer pH 7.0. Fractionation with PEG (molecular weight 6,000) in phosphate buffer was carried out exactly as with plasma at 21 C. and the fractions were spun down at 12,000 rpm. for 15 minutes. The bulk of the toxin was recovered at PEG concentrations between 7% and 9%.

It was further shown that this toxin retained its full toxicity after one months storage in the refrigerator. This illustrates that PEG which is a surface active agent, acts as a stabiliser for protein.

Example 3.Fractionation of botulinus toxin type D Fractionation was performed in the same manner as 7 before on a solution of one gm. of impure botulinus toxin type D in 100 ml. of phosphate butfer pH 7.0 at 21 C. and it was shown that the bulk of the active material was precipitated at PEG concentrations between 8% and 10%.

Example 4.Fractionation of tetanus antitoxin 100 ml. of immune horse serum 'was diluted with 100 ml. of M phosphate buffer pH 7.0 and fractionated with PEG molecular weight 6,000 in & M phosphate buffer as with plasma.

The gamma component was precipitated at 7% PEG concentration. The supernatant fluid after removal of the gamma component at 7% concentration was raised to 12% PEG concentration when the T component with approximately of gamma-globulin was recovered. This component contained 90% of the total activity of the original serum.

Example 5.Fractionation of pancreatic desoxyribonuelease Frozen bovine pancrease derived from Afrikander longhorn cattle is minced in a meat grinder and extracted for 24 hours with 0.25 N H 80 in a cold room. The extract is clarified and ammonium sulphate added to a concentration of and the precipitate discarded. The supernatant fluid is raised to concentration and the precipitate formed is redissolved in water and ammonium sulphate added to a concentration of 30%. 2 gms. of this precipitate are dialysed salt-free against very dilute H 80 pH 3.0, and diluted to ml. with H O.

At 15 C. 1 ml. of saturated ammonium sulphate is added to this solution and a portion of a 60% solution of PEG in H O, pH 3.0 (H 504), added to bring the concentration of PEG in the solution to 10%. The resulting precipitate is spun down, PEG solution is added to the supernatant fluid to a concentration of 20% and the resulting precipitate is spun down, and is purified desoxyribonuclease. The ratio AE260/ E280 (U.V. absorption), which is a measure of the activity and purity of the desoxyribonuolease was 0.10 in the original extract, and is 0.50, sometimes 0.80 in the purified product. During the processing the stabilising effect of PEG is very noticeable.

Example 7.Preparation of homogeneous gammaglobulin from blood plasma In the following preparation it is only desired to prepare gamma-globulin, and all other components of the original mixture (blood plasma) are to be discarded.

10 litres of plasma in citric acid solution (protein concentration 7%) are treated at 10 C. with 300 gms. sodium chloride and enough ammonium sulphate to raise the concentration from 0 to 1.8 molar in respect of ammonium sulphate. This treatment results in the precipitation of gamma-globulin in admixture with fibrinogen, beta-globulin and some other impurities whereas the major part of the albumin and some alpha-globulin remains in solution.

The precipitate is then redissolved in phosphate buffer (0.02 M, pH 7.0) to result in a solution having a 3% protein concentration (9,000 cc.). This solution is now fractionated at 18 C. by rapidly pouring into the solution polyethylene glycol (molecular weight 6,000) up to a total concentration of 14% PEG. Pouring rapidly in this context means as rapidly as is practical without entraining appreciable amounts of air bubbles. It is found that the total addition can be achieved within one minute. The mixture is left standing for approximately half an hour with gentle stirring (the optimum time may vary from batch to batch). The precipitate is separated by centrifugation and contains gamma-globulin in admixture with fibrinogen and traces of alpha-Z-globulin and beta-globulin. The solution which contains ammonium sulphate, some albumin and alpha-globulin is discarded.

The precipitate is redissolved in acetate buffer (pH 4.6, 0.02 M) to result in a protein concentration of 2.0% (9,000 cc.). This solution is fracionated at room temperature by the addition of polyethylene glycol in the same manner as above, except that the polyethylene glycol concentration is brought to 12% only and that the polyethylene glycol used is buffered with acetate buffer (pH 4.6, 0.02 M). Immediately after the addition of the polyethylene glycol the pH of the solution is adjusted, if necessary, with glacial acetic acid to pH 4.6. This time the precipitate, after having been removed by centrifugation is discarded. To the residual solution more polyethylene glycol (molecular weight 6,000) is added to raise the PEG content to 14%. The pH is also raised from 4.6 to between 6.3 and 6.5. This results in the precipitation of homogeneous gamma-globulin, which is separated by centrifugation.

For cases where traces of PEG in the final product are undesirable, the following additional step is added to the fractionating process:

The precipitate is dissolved in phosphate buffer (pH 7.0, 0.02 M) to result in a protein concentration of 3% (3,500 cc.), which solution is clarified by filtration. From this clarified solution the gamma-globulin may then be precipitated in a conventional manner at -8 C. with 25% ethanol. The precipitate is removed by centrifugation and vacuum dried. The dried gamma-globulin may then be dissolved in an 0.8% saline solution (using pyrogen-free Water) to result in a solution containing 16% of gamma-globulin. This solution is clarified, filtered and filled under sterile conditions into glass vials. A yield of 60% was obtainable.

All tests available indicated a very high degree of purity of the final product. The antibody content of the above product was compared with gamma-globulin prepared by a conventional method (MMED) by testing for the antibody content against the three polio vi'rus types. The antibody dilution end points determined for the two products were identical.

In a further test the purity of the gamma-globulin prepared by the above-described method was compared by free electrophoresis with a sample of commercial gammaglobulin prepared by cold ethanol fractionation. The same substances were also compared by ultracentrifugation. The gamma-globulin product prepared in accordance with the invention showed a single component only in both tests, Whereas the commercial gamma-globulin was found to be contaminated with approximately 3% of a substance having properties corresponding to those of normal human albumin and only contained 55% of substance having the actual properties of human gammaglobulin.

Mice were injected intraperitoneally with 0.5 ml. amounts of 16% gamma-globulin prepared by the abovedescribed method. No side effects could be observed.

Example 8.Preparation of pure fibrinogen and pure gamma-globulin from blood plasma 11 litres of human blood plasma (diluted with aqueous citric acid to a protein concentration of approximately 7%) are subjected to a preliminary fractionation at 10 C. by the addition of ammonium sulphate only to result in a concentration of 1.4 M ammonium sulphate. This results in a crude fibrinogen precipitate which is removed from the solution. The ammonium sulphate content of the remaining liquid is raised to 1.9 M, 330 gms. of sodium chloride are added and the temperature is raised to 18 C. The bulk of the gamma-globulin and some more fibrinogen is now precipitated in addition to other impurities, similar to those in the first precipitation step of Example 7. This crude gamma-globulin fraction is separated from the liquid and the liquid is dicarded.

The crude gamma-globulin precipitate is re-dissolved in phosphate bulfer (pH 7.0, 0.02 M) to result in a protein concentration of 2.5% (8,500 cc.). Polyethylene glycol (M. wt. 6,000) is added in the manner described in Example 7, while maintaining a temperature of 1 8 C. to give a polyethylene glycol concentration in the solution of 14%. The precipitate containing mainly gamma-globulin, some fibrinogen and some other impurities is now dissolved in acetate buffer (pH 4.6, 0.02 M) to result in a protein concentration of 2.4% (9,000 cc.) and polyethylene glycol is again added to the solution (at room temperature) to produce a concentration of 12.8% PEG. The PEG used is also buffered. The further procedure to obtain pure gamma-globulin follows the procedure described in Example 7.

The crude fibrinogen fraction obtained in the first precipitation step is dissolved in a citrate/saline buffer to result in a solution containing 1% protein (4,500 cc.) and having a pH of 7.0. Polyethylene glycol buffered with phosphate buffer (pH 7.0, 0.02 'M) is added to bring the PEG content of the solution to 4.5%, while working at room temperature (18 C.). The PEG is added in the same manner as described in Example 7 for the preparation of pure gamma-globulin. The precipitate is separated by centrifugation and found to be homogeneous fibrinogen containing some polyethylene glycol.

For further purification the fibrinogen precipitate is redissolved in citrate/saline buffer to result in a protein concentration of 1% (4,500 cc.) and centrifuged to remove denatured and insoluble material. From the clear solution pure fibrinogen is precipitated by adding ethanol to a concentration of 8% at -3 C. in a manner known as such. The precipitate is vacuum dried and may then be stored under sterile conditions in a manner known per se.

Fibrinogen prepared by the above-mentioned methods was found to be homogeneous apart from approximately 1% of macroglobulin which compares very favourably with a macroglobulin content of approximately 10% of macrog-lobulin found to be present in a comparative material produced by the known alcohol fractionation method.

Example 9.Purification of fibrinogen by extraction The crude fibrinogen precipitate obtained by the first fractionation step of Example 8 is mixed with kieselguhr and filled into a glass column. A solution of 4.5% PEG in water buffered with phosphate buffer (pH 7.0, 0.02 M) is prepared and slowly percolated through the column at room temperature. In this manner the impurities are selectively extracted from the column, leaving a purified fibrinogen behind on the column. In this procedure it is possible to achieve a fractionation of the impurities, should one so desire, by first fractionally eluting with stronger solutions of PEG progressively reducing the PEG concentration down to the final concentration of 4.5%.

Example l0.--Determination of the precipitation index As a measure of the comparative efllciency of a particular precipitant to precipitate a particular substance from a colloidal solution thereof, an arbitrary index, herein referred to as the precipitation index, may be conveniently used. This precipitation index is defined as the concentration of precipitant required to precipitate 50% of the substance to be precipitated from a solution containing 1 gm. per 100 ml. of the substance at a predetermined pH and temperature. In the present example it is assumed that the precipitation indices of polyethylene glycol samples of different molecular weight for gammaglobulin are to be determined. For this purpose a series of standard test solutions of the dilferent polymer samples are prepared in phosphate buffer of molarity of 0.066 and pH 7.2 to contain 2, 4, 6 n 2 gms. per 100 ml. of solution (n being any integer from 1 to 50).

ml. amounts of a 2% solution of gamma-globulin are placed in a series of tubes of a preparative ultracentrifuge. This is followed by the addition of 5 ml. of polymer solution of increasing concentration to the tubes. After thorough mixing the tubes are transferred to the rotor of the centrifuge, the temperature of which is maintained at 21 C. After allowing 30 minutes for the tubes to reach temperature equilibrium, the precipitates which form are removed by centrifugation at 12,000 rpm. for 15 minutes. The supernatant fluids are then carefully removed and their concentration determined by a standard biuret procedure.

Carrying out this test for samples of polyethylene gly- 'col of different average molecular weights (each sample being composed of molecules in a comparatively narrow molecular weight in the vicinity of 6,000 is considered Using PEG of molecular weight 300 the precipitation index is found to be just over 60. This can safely be considered a maximum acceptable value for all practical purposes. Using precipitants of higher molecular weight, it is found that the precipitation index initially drops very rapidly. For PEG of molecular weight 600 the index is just over 20. This corresponds to a quite convenient PEG concentration to work with, so that 600 may be considered as a more preferable lower limit for the molecular weight of the precipitant to be used. in the process in accordance with the present invention. From approximately a molecular weight of 1,500 onwards the effect of increasing the molecular weight on the precipitating index decreases very rapidly for which reason it is usually to be preferred to use a precipitant having a molecular weight from 1,500 upwards. In the case of PEG a molecular weight in the vicinity of 6,000 is considered the optimum because above that any advantage attainable in respect of an improved precipitating index is offset to an ever increasing extent by an increased viscosity which renders the substance less convenient to work with. Quite generally a molecular Weight of 100,000 can be considered the upper limit for practically useful precipitants and even above a molecular weight of 20,000 the high viscosity of the precipitants becomes seriously disadvantageous.

Example 11.Effect of precipitant on denaturisation of protein precipitate In some cases a change of the pH at which precipication is carried out helps to avoid denaturisation. Thus fibrinogen was observed to be denatured when precipi tated with PEG at pH 4.6 but not when precipitated at pH 6.3 to 7.0.

In the foregoing description and in the following claims due allowance is made for the difficulty of drawing a sharp distinction between true solution and colloidal dispersion applicable to all ranges of molecular weights of substances referred to in this specification. I shall therefore employ the term dispersion in the sense of a condition ranging from true solution to colloidal disperson.

It will be readily appreciated by those skilled in the art that the optimum conditions for any one separation problem can be readily predetermined by simple experiments in view of the foregoing teachings and can then be repeated over and again on a commercial scale by simple scaling up.

What I claim is:

1. A process for fractionating a mixture of proteinaceous substances which comprises:

(a) bringing said mixture into admixture with Water dispersible polyethylene glycol having a molecular weight substantially not less than 300 and substantially not more than 100,000, there being present also water in which said polyethylene glycol after said admixture is in a condition of dispersion ranging from true solution to colloidal dispersion, said polyethylene glycol-water dispersion being the only solvent phase present,

(b) adjusting the relative concentrations of the proteinaceous substances on the one hand and the remaining components in the product resulting from said admixture on the other hand to a predetermined value in dependency of pH and temperature at which value a part of said mixture is rendered indispersible in the water by the polyethylene glycol, resulting in the separation into a separate protein phase containing said part of said mixture and a separate aqueous liquid phase containing water and dispersed therein the remainder of said mixture with a composition difierent from the initial composition of said mixture,

(c) separating said separate protein phase as one fraction from said separate aqueous liquid phase as a second fraction; and

(d) recovering a proteinaceous fractionation product of said mixture from at least one of said fractions.

2. A process as claimed in claim 1 in which the polyethylene glycol used is selected with a molecular weight in the range of 1,500 to 20,000.

3. A process as claimed in claim 1 in which the mixture of proteinaceous substances is in aqueous dispersion prior to its admixture with polyethylene glycol and which comprises adding said polyethylene to the aqueous dispersion in an amount predetermined to precipitate the one fraction and retaining in aqueous dispersion the other fraction.

4. A process as claimed in claim 3 carried out at a temperature essentially above C.

5. A process as claimed in claim 3 in which the concentration of polyethylene glycol in admixture with the mixture being separated is successively increased thereby to precipitate successive fractions of different composition.

6. A process as claimed in claim 3 which comprises co-currently in -a predetermined ratio, bringing into intimate and uniform contact with one another two material streams, one being the mixture to be separated dispersed in water; and

the other being said polyethylene glycol.

7. A process as claimed in claim 3 which comprises carrying out the fractionation with at least two different precipitating agents in combination, of which at least one is an inorganic precipitant, capable as such of precipitating protein type substances from aqueous dispersion and one other is said polyethylene glycol.

8. A process as claimed in claim 7 in which the inorganic precipitant is ammonium sulphate.

9. A process as claimed in claim 8 applied to the separation of gamma-globulin from blood plasma, which comprises precipitating ran impure gamma-globulin from plasma by the addition thereto of ammonium sulphate, dissolving the precipitate in an aqueous medium and mixing the solution with polyethylene glycol to recover a more purified precipitate of gamma-globulin.

10. A process as claimed in claim 9 in which the lastmentioned precipitate is re-dissolved in an aqueous medium, contaminants of the gamma-globulin are precipitated by mixing the solution with a predetermined amount of polyethylene glycol, the precipitate is separated from the remaining solution and the concentration of polyethylene glycol in the solution is increased to precipitate gammaglobulin.

11. A process as claimed in claim 10 in which the removal of impurities at the lower concentration of polyethylene glycol takes place essentially at a pH of 4.4- 4.8, whereas the last-menti-oned precipitation of the gamma-globulin takes place essentially at a pH between 6 and neutral.

12. A process as claimed in claim 7 applied to the separation of both fibrinogen and gamma-globulin from blood plasma which comprises mixing the plasma with ammonium sulphate up to a concentration :at which most of the fibrinogen present precipitates, leaving the bulk of the gamma-globulin and some fibrinogen in solution, separating the precipitate from the solution, increasing the concentration of ammonium sulphate to precipitate at least the bulk of the gamma-globulin and re-dissolving both precipitates separately, mixing the redissolved first precipitate with polyethylene glycol to now precipitate an essentially pure fibrinogen fraction while maintaining essentially neutral conditions, and mixing the redissolved second precipitate with polyethylene glycol to now precipitate therefrom gamma-globulin in a more highly purified form than before.

13. A process as claimed in claim 1 which comprises:

(I) Bringing into intimate contact with one another:

(a) the mixture of proteinaceous substances in solid form; and

(b) a dispersion of said polyethylene glycol in water, the polyethylene glycol molecular weight, and the concentration pH and temperature of the dispersion being selected to cause a part of the mixture to become dispersed and to prevent another part of the mixture having a different composition, from becoming dispersed, and

(II) After a period of interaction between (a) and (b) separating a liquid phase containing the one said part dispersed therein, from the other undispersed part.

References Cited Partition of Cell Particles and Macromolecules, 1960, Stockholm and New York, Albertsson, pp. 8-11, 13-15, 27, 30-37, 77-80, 116-117, 123-144, 146-148, 171-172 and 223-226.

Biochimica et Biophysica Acta, vol. 69, pages 263-270, July 1962, Lentz et al. Biochimica et Biophsica Acta, vol. 82, pages 463-471 and 474-475, Polson et al.

WILLIAM H. SHORT, Primary Examiner. HOWARD SCHAIN, Assistant Examiner.

US. Cl. X.R. 

